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1.
Gastroenterology ; 147(4): 822-834.e13, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25038433

RESUMO

BACKGROUND & AIMS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS: We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS: Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS: ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Intestinos/enzimologia , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Nicho de Células-Tronco , Células-Tronco/enzimologia , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Células Enteroendócrinas/enzimologia , Células Caliciformes/enzimologia , Intestinos/citologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Organoides , Celulas de Paneth/enzimologia , Fenótipo , Transdução de Sinais , Fatores de Tempo
2.
Cancer Res ; 73(20): 6359-74, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24097820

RESUMO

Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the stepwise progression of the human disease. The inflammatory cytokine interleukin (IL)-6 is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL-6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL-6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL-6 synergizes with oncogenic Kras to activate the reactive oxygen species detoxification program downstream of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling cascade. In addition, IL-6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL-6 emerges as a key player at all stages of pancreatic carcinogenesis and a potential therapeutic target.


Assuntos
Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/fisiologia , Neoplasias Pancreáticas/metabolismo , Animais , Progressão da Doença , Humanos , Interleucina-6/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Estresse Oxidativo/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Distribuição Aleatória
3.
J Clin Invest ; 122(2): 639-53, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22232209

RESUMO

Pancreatic cancer is almost invariably associated with mutations in the KRAS gene, most commonly KRASG12D, that result in a dominant-active form of the KRAS GTPase. However, how KRAS mutations promote pancreatic carcinogenesis is not fully understood, and whether oncogenic KRAS is required for the maintenance of pancreatic cancer has not been established. To address these questions, we generated two mouse models of pancreatic tumorigenesis: mice transgenic for inducible KrasG12D, which allows for inducible, pancreas-specific, and reversible expression of the oncogenic KrasG12D, with or without inactivation of one allele of the tumor suppressor gene p53. Here, we report that, early in tumorigenesis, induction of oncogenic KrasG12D reversibly altered normal epithelial differentiation following tissue damage, leading to precancerous lesions. Inactivation of KrasG12D in established precursor lesions and during progression to cancer led to regression of the lesions, indicating that KrasG12D was required for tumor cell survival. Strikingly, during all stages of carcinogenesis, KrasG12D upregulated Hedgehog signaling, inflammatory pathways, and several pathways known to mediate paracrine interactions between epithelial cells and their surrounding microenvironment, thus promoting formation and maintenance of the fibroinflammatory stroma that plays a pivotal role in pancreatic cancer. Our data establish that epithelial KrasG12D influences multiple cell types to drive pancreatic tumorigenesis and is essential for tumor maintenance. They also strongly support the notion that inhibiting KrasG12D, or its downstream effectors, could provide a new approach for the treatment of pancreatic cancer.


Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Diferenciação Celular , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Pancreatite/patologia , Pancreatite/fisiopatologia , Transdução de Sinais/fisiologia , Taxa de Sobrevida
4.
Prostate ; 72(2): 217-24, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21594883

RESUMO

BACKGROUND: Loss-of-function of Pax2 results in severe defects of the male reproductive system, and Pax2 expression is detected in mouse prostate lobes and human prostatic cancers. However, the role for Pax2 in prostate development remains poorly understood. METHODS: The expression of Pax2 was examined by in situ hybridization at various developmental stages. Urogenital sinuses were dissected out at E18.5 from mouse Pax2 mutants and controls, cultured in vitro or grafted under the renal capsule of CD1 nude mice. The expression of prostate developmental regulatory factors was analyzed by semi-quantitative real-time PCR or immuohistochemistry. RESULTS: Pax2 is expressed in the epithelial cells of prostate buds. Loss-of-function of Pax2 does not affect the initiation of prostatic buds, but in vitro culture assays show that the prostates of Pax2 mutants are hypomorphic and branching is severely disrupted compared to controls. RT-PCR data from Pax2 mutant prostates demonstrate increased expression levels of dorsolateral prostate marker MSMB and ventral prostate marker SBP and dramatically reduced expression levels of anterior prostate marker TGM4. CONCLUSIONS: Pax2 is essential for mouse prostate development and regulates prostatic ductal growth, branching, and lobe-specific identity. These findings are important for understanding the molecular regulatory mechanisms in prostate development.


Assuntos
Fator de Transcrição PAX2/fisiologia , Próstata/fisiologia , Animais , Glicoproteínas/genética , Glicoproteínas/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Nus , Morfogênese/fisiologia , Oxirredutases , Fator de Transcrição PAX2/genética , Próstata/embriologia , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transglutaminases/genética , Transglutaminases/metabolismo
5.
Dev Dyn ; 237(11): 3410-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18942146

RESUMO

Hox genes are crucial for body axis specification during embryonic development. Hoxa11 plays a role in anteroposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximodistal patterning of the limbs. Hoxa11 expression is also observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock-in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in-frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis.


Assuntos
Alelos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Homeodomínio/biossíntese , Organogênese/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Transgênicos , Tubo Neural/embriologia , Especificidade de Órgãos/fisiologia , Proteínas Recombinantes de Fusão/genética , Sistema Urogenital/embriologia
6.
Development ; 134(16): 2981-9, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17626057

RESUMO

Unlike the rest of the axial skeleton, which develops solely from somitic mesoderm, patterning of the rib cage is complicated by its derivation from two distinct tissues. The thoracic skeleton is derived from both somitic mesoderm, which forms the vertebral bodies and ribs, and from lateral plate mesoderm, which forms the sternum. By generating mouse mutants in Hox5, Hox6 and Hox9 paralogous group genes, along with a dissection of the Hox10 and Hox11 group mutants, several important conclusions regarding the nature of the ;Hox code' in rib cage and axial skeleton development are revealed. First, axial patterning is consistently coded by the unique and redundant functions of Hox paralogous groups throughout the axial skeleton. Loss of paralogous function leads to anterior homeotic transformations of colinear regions throughout the somite-derived axial skeleton. In the thoracic region, Hox genes pattern the lateral plate-derived sternum in a non-colinear manner, independent from the patterning of the somite-derived vertebrae and vertebral ribs. Finally, between adjacent sets of paralogous mutants, the regions of vertebral phenotypes overlap considerably; however, each paralogous group imparts unique morphologies within these regions. In all cases examined, the next-most posterior Hox paralogous group does not prevent the function of the more-anterior Hox group in axial patterning. Thus, the ;Hox code' in somitic mesoderm is the result of the distinct, graded effects of two or more Hox paralogous groups functioning in any anteroposterior location.


Assuntos
Osso e Ossos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Costelas/embriologia , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Osso e Ossos/metabolismo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Camundongos , Modelos Biológicos , Costelas/metabolismo , Distribuição Tecidual
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